Expression and purification of recombinant polypeptides and proteins is a routine process within biotechnological research. In general the process of purification comprises the expression of a desired polypeptide in prokaryotic or eukaryotic cells followed by the separation from other non-proteinacious and proteinacious particles of the host cell. Thereby various types of chromatography are used to purify the desired molecule e.g. by size, charge or hydrophobicity.
One further specific strategy is to use a tag which is fused to the polypeptide of interest. Specific tags can be used to support the folding, solubility, stability and expression of the polypeptide of interest while other tags are mainly used for purification. Thereby the desired polypeptide is expressed as a fusion construct in prokaryotic or eukaryotic cells and can be purified via the fused tag which is detected by a specific antigen binding moiety. This kind of purification strategy is called affinity chromatography.
One purification-tag used in the scientific community is e.g. the His-tag. Thereby the polypeptides which are fused with a His-tag can be separated by using e.g. a purification column with immobilized nickel or cobalt ions that have strong affinity to the His-tag. The protein is then released from the column in an elution process involving imidazole which competes with the His-tags for nickel or cobalt binding. Further examples are the Flag-tag and the Strep-tag which are both fused to the polypeptide of interest and serve as an antigen for respective tag-specific antigen binding moieties like e.g. antibodies or Streptactin, respectively. These binding moieties (e.g. antibodies, streptactin or metal ions) which are used for purification (e.g. via the Flag-tag, Strep-tag or His-tag, respectively) can e.g. be immobilized on a solid substrate (e.g. membranes, beads). Those solid substrates coupled with specific binding moieties for defined tags can be used to easily capture the tagged polypeptide from complex samples as lysates or conditioned media. However, the Flag-, Strep- and His-tags which are short peptides are sometimes not accessible within the 3-dimensional structure of specific polypeptides or proteins and thus not suitable for purification. Additionally, purification from mammalian cell culture supernatants via the Strep-tag is impaired due to the high biotin concentrations of most media.
Certain larger globular tags can support the folding, solubility and expression of difficult-to-express polypeptides as proteins. Most available gene-fusion-technologies were developed for expression in E. coli and purification from crude lysates. Examples of those fusion proteins are MBP (Maltose binding protein), GST (Glutathione-S-Transferase) and SUMO (small ubiquitin modifying protein; see for example WO 03/057174).
The SUMO-tag has originally been designed for prokaryotic expression (e.g. SUMOpro™ Expression Kit, www.lifesensors.com), and was then further developed for mammalian expression (SUMOstar™ Expression Kit, www.lifesensors.com). SUMO functions both as a chaperon and as an initiator of protein folding to improve the solubility and level of expression of the protein of interest. By using a desumoylase, the SUMO tag, fused to the N-terminus of the protein of interest, can be removed resulting in the production of native N-terminus of the protein. Fusion of SUMO tag to the C-terminus of the protein of interest does not allow the removal of the fusion tag. Purification of the target protein fused to SUMO tag does not utilize the SUMO tag but requires the application of a purification tag such as His-tag.
An alternative for mammalian expression is the usage of the Fc-tag which comprises the hinge-region, the CH2 and CH3 domain of the human IgG1. The Fc-tag is used to support expression, folding and secretion of specific polypeptides and in parallel is also used as a tag for its purification. While the His- and the Flag-tag are short peptides with low molecular weight and well suited for the expression of soluble polypeptides and proteins, the Fc-tag is a polypeptide of more than 200 amino acids and supports the expression of specific hydrophobic less-soluble proteins. However, the relatively large Fc-portion forms disulfide-bridged aggregates, resulting in dimeric or multimeric forms of the isolated and purified protein of interest.
Other common alternatives are the GST (glutathione S-transferase) and MBP (maltose binding protein), which bind to glutathione and maltose, respectively. Both tags are of high molecular weight (>25 kDa) and significantly increase the solubility and stability of a polypeptide or protein of interest. However, both gene-fusion systems cannot be used for protein purification of secreted proteins from conditioned mammalian cell culture supernatants as ingredients of the media prevent binding of the fusion tag to its binding partner, i.e. glutathione or maltose. Additionally, both fusion tags have a tendency to aggregate in mammalian expression systems and also tend to form inclusion bodies.
Hence, while e.g. the Fc-Tag, is not suited for the expression and purification of monomeric polypeptides and proteins, all other available tags have specific assets and drawbacks and are not suited for the expression and/or purification of certain specific polypeptides or proteins. Taken together, the quality of expression and purification not only depends on the nature of the polypeptide or protein of interest but also on the respective tag that is used. Thus the combination of a specific tag and a specific polypeptide or protein of interest is crucial for best results but hardly predictable. Consequently, there is an inexhaustible need for novel and convenient tags that enable expression and purification or improve quality of specific challenging recombinant polypeptides and proteins. The methods disclosed in the present application provide an efficient way to express and purify polypeptide or protein by using lysozyme as a tag.